title: Homogeneous immunosubtraction integrated with sample preparation enabled by a microfluidic format.
abstract: Immunosubtraction is a powerful and resource-intensive laboratory medicine assay that reports both protein mobility and binding specificity. To expedite and automate this electrophoretic assay, we report on advances to the electrophoretic immunosubtraction assay by introducing a homogeneous, not heterogeneous, format with integrated sample preparation. To accomplish homogeneous immunosubtraction, a step-decrease in separation matrix pore-size at the head of a polyacrylamide gel electrophoresis (PAGE) separation channel enables "subtraction" of target analyte when capture antibody is present (as the large immune-complex is excluded from PAGE), but no subtraction when capture antibody is absent. Inclusion of sample preparation functionality via small pore size polyacrylamide membranes is also key to automated operation (i.e., sample enrichment, fluorescence sample labeling, and mixing of sample with free capture antibody). Homogeneous sample preparation and assay operation allows on-the-fly, integrated subtraction of one to multiple protein targets and reuse of each device. Optimization of the assay is detailed which allowed for ~95% subtraction of target with 20% non-specific extraction of large species at the optimal antibody-antigen ratio, providing conditions needed for selective target identification. We demonstrate the assay on putative markers of injury and inflammation in cerebrospinal fluid (CSF), an emerging area of diagnostics research, by rapidly reporting protein mobility and binding specificity within the sample matrix. We simultaneously detect S100B and C-reactive protein, suspected biomarkers for traumatic brain injury (TBI), in ~2 min. Lastly, we demonstrate S100B detection (65 nM) in raw human CSF with an estimated lower limit of detection of 3.25 nM, within the clinically relevant concentration range for detecting TBI in CSF. Beyond the novel CSF assay introduced here, a fully automated immunosubtraction assay would impact a spectrum of routine but labor and time-intensive laboratory medicine assays.
authors.affiliation: Department of Bioengineering, University of California Berkeley, Berkeley, California 94720, USA.
authors.forename: Akwasi A
authors.initials: AA
authors.lastname: Apori
authors.forename: Amy E
authors.initials: AE
authors.lastname: Herr
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000295:instrumentation*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015415:Biomarkers / Q000134:cerebrospinal fluid; D001930:Brain Injuries / Q000134:cerebrospinal fluid; D002556:Cerebrospinal Fluid Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D004586:Electrophoresis; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D046210:Microfluidic Analytical Techniques; D009414:Nerve Growth Factors / Q000134:cerebrospinal fluid / Q000276:immunology / Q000302:isolation & purification; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000134:cerebrospinal fluid / Q000276:immunology / Q000302:isolation & purification; D013379:Substrate Specificity; D018511:Systems Integration
chemical_list: D000906:Antibodies; D015415:Biomarkers; D002556:Cerebrospinal Fluid Proteins; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human